Immunizing against hiv infection

ABSTRACT

A virus neutralizing level of antibodies to a primary HIV isolate is generated in a host by a prime-boost administration of antigents. The primary antigen is a DNA molecule encoding an envelop glycoprotein of a primary isolate of HIV-1 while the boosting antigen is either a non-infectious, non-replicating HIV-like particle having the envelope glycoprotein of a primary isolate of HIV-1 or an attenuated viral vector expressing an envelope glycoprotein of a primary isolate of HIV-1.

FIELD OF THE INVENTION

[0001] The present invention relates to the field of immunology and, in particular, to methods and compositions for immunizing a host against infection with HIV.

BACKGROUND OF THE INVENTION

[0002] Human immunodeficiency virus is a human retrovirus and is the etiological agent of acquired immunodeficiency syndrome (AIDS). It is estimated that more than 33 million people have been infected with HIV world-wide as of December 1999 (Ref 1—various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure).

[0003] As the HIV epidemic continues to spread world wide, the need for an effective vaccine remains urgent. Efforts to develop such a vaccine have been hampered by several factors three of which are: (a) the extraordinary ability of the virus to mutate; (b) inability of most known specificities of anti-HIV antibodies to neutralise HIV primary isolates consistently; and (c) lack of understanding of the correlates of protective immunity to HIV infection. Over the last 10 years, several candidate HIV vaccines have been tested in primates for their immunoprotective abilities (Ref 2). These studies suggest that both neutralising antibodies and cell-mediated immunity play a role in conferring sterilizing immunity and preventing progression towards disease (Ref 3, 4). While the correlates for immune protection against HIV-1 infection are currently unknown, an effective HIV vaccine should elicit both strong neutralising antibody and cytotoxic T lymphocyte (CTL) responses.

[0004] Envelope subunit vaccines have been shown to induce high titred humoral responses, but were inefficient in eliciting CTL responses (Ref 5). Live recombinant pox vectors have been shown to elicit very potent CTL responses, however these vectors were ineffective for generating a significant antibody response (Ref 6). In attempts to combine the two immunization types, several clinical trials involved a prime-boost strategy, consisting of initial viral vector immunization followed by boosts with recombinant HIV-1 envelope subunits (Ref 7, 8), have led to limited success with respect to CTL responses. Other vaccine approaches have used non-infectious, non-replicating, immunogenic virus-like particles (VLP) for immunising against HIV infection (Ref 9, 10). This type of immunogen has lead to the generation of neutralizing antibodies to a laboratory HIV-1 strain (Ref 10).

[0005] A prime-boost approach has been investigated using non-infectious VLPs to enhance HIV-specific CTL responses in mice primed with recombinant canarypox vector vCP205 encoding HIV-1 gp 120 (MN strain) (Ref 11). This study showed that VLPs could boost the CTL response to the canarypox vector.

[0006] Recently, a study showing the induction of neutralizing antibodies to a HIV-1 primary isolate in chimpanzees has been reported (Ref 12). In this study, recombinant adenovirus expressing gp160 was used as the priming agent and recombinant gp120 protein was used to boost the monkeys.

[0007] There is still a need for vaccines and immunization regimes to induce both a strong CTL response as well as neutralizing antibodies to HIV primary isolates.

SUMMARY OF THE INVENTION

[0008] In accordance with one aspect of the present invention, there is provided a method for generating, in a host, particularly a human host, a virus neutralizing level of antibodies to a primary HIV isolate, comprising at least one administration of a priming antigen to the host, wherein the priming antigen comprises a DNA molecule encoding an envelope glycoprotein of a primary isolate of HIV, resting the host for at least one specific resting period to provide for clonal expansion of an HIV antigen specific population of precursor B-cells therein to provide a primed host, and at least one administration of a boosting antigen to the primed host to provide said neutralizing levels of antibodies, wherein the boosting antigen is selected from the group consisting of a non-infectious, non-replicating, immunogenic HIV-like particle having at least part of the envelope glycoprotein of a primary isolate of HIV and an attenuated viral vector expressing at least part of an envelope glycoprotein of a primary isolate of HIV.

[0009] The primary HIV isolate may be an HIV-1 isolate including from the clade B HIV-1 clinical isolate HIV-1_(Bx08), although any other primary HIV-1 isolate may be employed in the immunization procedures of the invention.

[0010] The DNA molecule encoding the envelope glycoprotein of a primary isolate of HIV may be contained in a plasmid vector under the control of a heterologous promoter, preferably a cytomegalovirus promoter, for expression of the envelope glycoprotein in the host, which may be a human host.

[0011] The vector utilized for DNA molecule immunization is novel and constitutes a further aspect of the present invention. Preferably, the vector has the identifying characteristics of pCMV3Bx08 shown in FIG. 2, such identifying characteristics being the nucleic acid segments and restriction sites identified in FIG. 2.

[0012] A priming administration of antigen may be effected in a single or in multiple administrations of the priming antigen. In the latter case, the at least one specific resting period to permit clonal expression of HIV antigen-specific population precursor B-cells may be effected after each priming administration. The at least one specific resting period may be between about 2 and 12 about months.

[0013] In the embodiment where the boosting antigen is a non-infectious, non-replicating, immunogenic HIV-like particle, such particle may comprise an assembly of:

[0014] (i) an env gene product,

[0015] (ii) a pol gene product, and

[0016] (iii) a gag gene product

[0017] with the particle being encoded by a modified HIV genome deficient in long terminal repeats (LTRs) and containing gag, pol and env in their natural genomic arrangement. Such particles and the manufacture thereof are described in U.S. Pat. No. 5,439,809, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference. Such particles can include mutations in gag and pol to further reduce potential infectivity, as more fully described in U.S. Pat. No. 6,080,408, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference (WO 96/06177). In a preferred embodiment, the env gene is that from primary isolate BX08. The gag gene and pol gene may be those from the same primary isolate or may be chosen from those of other HIV-1 isolates, which maybe primary isolates.

[0018] The non-infectious, non-replicating, immunogenic HIV-like particle may be administered in conjunction with an adjuvant. Any suitable adjuvant may be used, such as QS21, DC-chol, RIBI or Alum.

[0019] Such non-infectious, non-replicating, immunogenic HIV particle may be formed by expression from a suitable vector in mammalian cells. In accordance with an additional aspect of this invention, there is provided a vector comprising a modified HIV-genome deficient in long terminal repeats and a heterologous promoter operatively connected to said genome for expression of said genome in mammalian cells to produce the non-infectious, non-replicating and immunogenic particle, wherein at least the env gene of the modified HIV-genome is that from a primary isolate of HIV. The gag and pol genes of the modified HIV genome may be those from the same primary isolate or those from another isolate, which may be a primary isolate.

[0020] The vector preferably is a plasmid vector while the primary isolate preferably is BX08. The promoter may be the metallothionein promoter. The vector preferably has the identifying characteristics of plasmid p133B1 shown in FIG. 3, such identifying characteristics being the nucleotide segments and restriction sites identified in FIG. 3.

[0021] In the embodiment where the boosting antigen is an attenuated viral vector, the attenuated viral vector may be an attenuated avipox virus vector, particularly the attenuated canary poxvirus ALVAC. The attenuated viral vectors used herein form another aspect of the invention. The attenuated viral vector may contain a modified HIV genome deficient in long terminal repeats (LTRs), wherein at least the env gene is that from primary isolate BX08. The gag and pol genes of the modified genome may be those from the same primary isolate or may be chosen from other HIV isolate.

[0022] The attenuated canarypox virus-based vector ALVAC is a plaque-cloned derivative of the licensed canarypox vaccine, Kanapox, and is described in reference 19. The attenuated canary pox vector preferably has the identifying characteristics of vCP1579 shown in FIG. 4, such identifying characteristics being the nucleic acid segments and restriction sites identified in FIG. 4.

[0023] The at least one administration of a boosting antigen may be effected in a single administration or at least two administration of the boosting antigen.

[0024] The invention further includes compositions comprising the immunogens as provided herein and their use in the manufacture and formulation of immunogenic compositions including vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further understood from the following description with reference to the drawings, in which:

[0026]FIG. 1 shows the details of the elements of plasmid pCMVgDtat⁻ vpr⁻Bx08.

[0027]FIG. 2 shows the details of the elements of plasmid pCMV3Bx08.

[0028]FIG. 3 shows the details of the elements of plasmid p133B1.

[0029]FIG. 4 shows the details of the insertions into ALVAC (2) to provide vector vCP1579.

[0030]FIGS. 5A and 5B contain a representation in time-line form of the immunization regime used wherein the study groups are described in Table 1. The numbers below the lines refer to weeks.

[0031]FIG. 6 shows the immunoreactivity to HIV-1 antigens of the serum diluted 1:100 from the macaques immunized with the various preparations as described in Table 1.

[0032]FIG. 7 shows the immunoreactivity to HIV-1 antigens of the serum diluted 1:1000 from the macaques immunized with the various preparations as described in Table 1.

[0033]FIG. 8 shows the details of the elements of pMPC6H6K3E3.

[0034]FIG. 9 shows the details of the elements of pMPC5H6PN.

[0035]FIG. 10 shows the details of the elements of pHIV76.

[0036]FIG. 11 shows the nucleotide sequence (SEQ ID NO: 1) for the H6/HIV Pol/Nef epitope cassette in the ALVAC C5 site of vCP1579.

[0037]FIG. 12 contains the nucleotide sequence of C6 region (coding strand SEQ ID NO: 16, complementary strand SEQ ID NO: 17, K3L amino acid sequence SEQ ID NO: 18, E3L amino acid sequence SEQ ID NO: 19).

GENERAL DESCRIPTION OF INVENTION

[0038] As noted earlier, the present invention involves administration of HIV antigens to elicit virus-neutralizing levels of antibodies against a primary HIV isolate.

[0039] A DNA construct was prepared incorporating the env gene from the primary isolate Bx08 under the control of the cytomegalovirus promoter and the construct, pCMV3Bx08, is shown in FIG. 2. The construct pCMV3Bx08 is derived from plasmid pCMVgDtat⁻vpr⁻Bx08 seen in FIG. 1. The DNA construct pCMV3Bx08 was used in a priming immunization step to a host, macaque monkeys being the animal model chosen.

[0040] Following the priming immunization step, which may be effected in one or more administrations of the DNA construct, the host is allowed to rest to provide for clonal expression of an HIV antigen specific population of precursor B-cells therein to provide a primed host.

[0041] The boosting administration is effected either with a non-infectious, non-replicating, immunogenic HIV-like particle (VLP) or an attenuated viral vector.

[0042] For this purpose, a VLP expression plasmid was constructed containing a modified HIV genome lacking long terminal repeats in which the env gene is derived from primary isolate BX08, wherein the modified HIV genome is under the control of a metallothionein promoter. The construct, p133B1, shown in FIG. 3, was used to effect expression in mammalian cells of the non-infectious, non-replicating, immunogenic HIV-like particules, in which the env gene product is that from the primary isolate BX08.

[0043] In the case of the attenuated virus vector, a recombinant attenuated canarypox virus vector was constructed to contain the env gene from primary isolate BX08. The viral vector vCP1579 (FIG. 4) was prepared by a variety of manipulations from plasmid pHIV76 (FIG. 10), as shown described in detail below.

[0044] These products were utilized in a boosting administration to the primed macaques. The boosting administration may be effected in one or more immunizations. In a preferred aspect of the invention, the non-infectious, non-replicating immunogenic HIV-like particles may co-administered with the DNA construct in the priming administration and the DNA construct may be coadministered with the HIV-like particles in the boosting administration.

[0045] Immunizations were effected in accordance with the procedure of the invention and the results obtained were compared with those obtained using other protocols according to the protocols set forth in Table 1. The immunization regimes used are shown as time lines in FIGS. 5A and 5B.

[0046] The results obtained following the various protocols showed that, in particular, a primary DNA vaccination in combination with a boost from either the VLP or the attenuated canarypox virus enhanced the levels of neutralizing antibodies, as indicated by the reduction of detectable p24 levels in cells infected with primary HIV isolates.

[0047] Biological Deposits

[0048] Certain vectors that are described and referred to herein have been deposited with the American Type Culture Collection (ATCC) located at 10801 University Boulevard Manassas, Va. 20110-2209, USA, pursuant the Budapest Treaty and prior to the filing of this application. Samples of the deposited vectors will become available to the public and all restrictions imposed or access to the deposits will be received upon grant of a patent based on this United States patent application or the United States patent application in which they are described. In addition, the deposit will be replaced if viable samples cannot be dispensed by the Depository. The invention described and claimed herein is not limited in scope by the biological materials deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent of similar vectors that contain nucleic acids which encode equivalent or similar antigens as described in this application are within the scope of the invention. Deposit Summary Plasmid ATCC Deposit Date pMT-HIV  40912 Oct. 12, 1990 pCMVgDtat{overscore ( )}vpr{overscore ( )} 209446 Nov. 11, 1997

EXAMPLES

[0049] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

Example 1

[0050] This Example describes the construction of plasmid pCMV3BX08.

[0051] The plasmid, pCMV3BX08, contains sequence segments from various sources and the elements of construction are depicted in FIG. 2.

[0052] The prokaryotic vector pBluescript SK (Stratagene) is the backbone of the plasmid pCMV3.BX08 and was modified by the replacement of the Amp^(R) with Kan^(R) gene and the deletion of the fl and the LacZ region. To achieve the desired modifications, the sequence between Ahd1 (nucleotide 2,041) and Sac1 (nucleotide 759) of pBluescript SK, which contains the Amp^(R), fl origin and the LacZ, was deleted. A 1.2 kb Pst1 fragment from the plasmid pUC-4K (Pharmacia) containing the Kan^(R) gene, was blunt end ligated to the Ahd1 site of pBluescript SK in a counter-clockwise orientation relative to it's transcription. A 1.6 kb Ssp1/Pst1 DNA fragment containing the human cytomegalovirus immediate-early gene promotor, enhancer and intron A sequences (CMV) was ligated to the other end of the Kan^(R) gene so that the transcription from the CMV promoter proceeds in the clockwise orientation. A synthetic oligonucleotide segment containing translation initiation sequence and sequences encoding the human tissue plasminogen activator signal peptide (TPA) was used to link the CMV promotor and the sequences encoding the envelope gene of the primary isolate HIV-1_(BX08).

[0053] The envelope gene from the HIV-1 primary isolate BX08 was isolated from the plasmid pCMVgDtat⁻vpr⁻Bx08 illustrated in FIG. 1. The plasmid pCMVgDtat⁻vpr⁻Bx08 was derived from the deposited plasmid pCMVgDtat⁻vpr⁻, the construction of which is described in copending U.S. patent application Ser. No. 08/991,773 filed Dec. 16, 1997, assigned to the assignee hereof and the disclosure of which is incorporated herein by reference, (WO 99/31250). The plasmid pCMVgDtat⁻vpr⁻Bx08 was derived by substituting the BX08 envelope sequence from clade B HIV-1 clinical isolate HIV-1_(BX08) for the modified HIV genome sequence present in pCMVgDtat⁻vpr⁻. Plasmid pCMVgDtat⁻vpr³¹Bx08 was restricted with the restriction enzyme Xho I and made blunt ended with Klenow treatment. A Not I partial digestion was then performed and the resulting 6.3 kb fragment containing the env gene was isolated. Plasmid pCMV3 (Invitrogen) was restricted with Bam HI and made blunt ended with Klenow treatment. The plasmid pCMV3 was then restricted with Not I and the resulting 4.4 kb fragment was isolated. The 6.3 and 4.4 kb fragments were ligated together to produce plasmid pCMV3BX08 (FIG. 2).

[0054] The pCMV3BX08 construct was introduced into HB101 competent cells according to manufacturer's recommendations (GibcoBRL). Correct molecular clones were identified by restriction and sequencing analysis and their expression of envelope glycoprotein was examined in transient transfections followed by Western blot analysis.

[0055] All DNAs used for immunizations were prepared using EndoFree Plasmid Kit (Qiagen). For intramuscular immunizations either 3 mg or 600 μg of pCMVBX08, in 100 μl PBS was injected.

[0056] Proviral DNA for clade B HIV-1 clinical isolate HIV-1_(BX08) originated at Transgene (Strasbourg, France) and was isolated from genomic DNA of cells infected with the virus.

Example 2

[0057] This Example describes the construction of plasmid p133B1.

[0058] A Bx08 plasmid expression vector (p133B1, FIG. 3) used to transfect the mammalian cells was engineered in several stages using pUC18 as the initial host plasmid. First, an 8.3-kbp fragment of HIV-1_(LAI) provirus encoding the gag, pol and env proteins was isolated. This fragment lacked the transcription regulatory elements and long terminal repeat elements from each end of the proviral genome to ensure the virus-like particles would be replication-incompetent. This fragment was linked to an inducible human type IIA metallothionein (MTIIA) promoter (Ref 13) and also to a Simian Virus 40 polyadenylation (polyA) addition/transcription termination sequence from plasmid pSV2dhfr (Ref 14). The modified fragment was then inserted into the pUC18 host vector. The resulting deposites expression construct, named pMT-HIV, was used to transfect into African green monkey kidney (Vero) and COS monkey kidney cells. The procedure for obtaining pMT-HIV is further described in the aforementioned U.S. Pat. No. 5,439,809. Both transfected cell lines produced non-replicating virus-like particles when induced with metal ions (Ref 15).

[0059] Two further modifications were made to the proviral DNA in pMT-HIV to provide additional safety features to protect human cells against recombination events with reverse-transcribed DNA:

[0060] 1) inactivation of the RNA packaging sequences; and

[0061] 2) deletion of a large section of the pol gene encoding reverse transcriptase and integrase.

[0062] To delete the first RNA packaging signal, part of the DNA corresponding to the untranslated leader sequence of the mRNA was replaced with synthetic DNA lacking a 25-bp motif corresponding to nucleotides 753-777 (the psi sequence). To inactivate the second RNA packaging signal, two adenosine residues within a gag gene zinc finger sequence were changed to thymidine residues. Each of these residue changes had the effect of replacing cysteine residues in a Cys-His array with a serine in the gene product.

[0063] The pol gene deletion was effected by replacing a 1.9-kbp fragment with synthetic DNA containing stop codons in all three reading frames. This prevented read-through translation of the residual integrase coding sequence on the 3′ side of the deletion. The 1.9-kbp deletion in pol also eliminated the expression of reverse transcriptase and integrase enzymes. However, the deletion left intact the gene encoding the viral protease, which is both an immunogenic component of HIV-1 virus particles and allows the expression of particles with processed gag antigens closely resembling native virions (Ref 16). The protease also contains epitopes that are conserved across HIV-1 clades. The modifications described with respect to gag and pol genes are more fully described in the aforementioned U.S. Pat. No. 6,080,408 (WO 96/06177).

[0064] Finally, the HIV-1_(LAI) env gene within pMT-HIV was replaced with that of HIV-1_(Bx)08. To effect this replacement, a 2440-bp fragment containing the env gene of Bx08 was amplified by polymerase chain reaction (PCR) from cells infected with this isolate. The PCR product was then used to replace the corresponding region present in pMT-HIV. However, the incoming fragment from HIV-1_(Bx)08 was 125-bp shorter than the original HIV-1_(LAI) region owing to a deletion in the untranslated region between the env gene stop codon and the termination/polyA addition sequence. The resulting construct replaced all but eleven amino acid residues of the LAI envelope proteins gp120 and gp41. Of these eleven, only the first three differ between the LAI and Bx08 isolates, and these are all charge-conservative changes meaning the final expression vector (p133B1) encoded a nearly authentic HIV-1_(Bx08) env protein.

Example 3

[0065] This Example describes the production of HIV-like particles.

[0066] African green monkey kidney (Vero) cells were recovered and cultivated in Dulbecco's modified Eagle medium (DMEM) containing 10% v/v fetal bovine serum (FBS), referred to below as Complete Medium. At passage 141, the cells were transfected with p133B1 using the calcium phosphate method when at approximately 30% confluence. The cells were shocked with glycerol 8 hours after transfection. For this step, six 10-cm dishes containing approximately 3.0×10⁶ cells each in 10.0 mL of Complete Medium were prepared. Each dish received 25.0 μg of expression vector and 2.0 μg of plasmid pSV2neo (Ref 17). The pSV2neo contains a selectable marker gene conferring resistance to the antibiotic geneticin (G418). Two days after transfection, the cells from each dish were recovered by trypsinization and replated into twenty-five fresh dishes in Complete Medium supplemented with 0.5 mg/mL of G418.

[0067] In total, 394 colonies were isolated from the dishes using cloning cylinders. Each colony was recovered by trypsinization and divided into two cluster dish wells, one of the wells per clone was induced after reaching 50% to 90% confluence. Prior to induction, the wells were treated by replacing all the medium with fresh Complete Medium containing 10.0 μM 5-azacytidine. After incubating for between 18 hours and 22 hours, the medium was removed and replaced with fresh DMEM containing 0.2% v/v FBS, 2.0 μM CdCl₂ and 200.0 μM ZnCl₂. The wells were incubated for a further 20 hours to 24 hours at which time samples of the medium were removed and tested by p24 ELISA.

[0068] The twenty highest-producing clones, based on the p24 titre, were chosen and cells from the corresponding uninduced wells were sub-cultured into one T-25 and one T-150 flask per clone. Both flasks were grown to confluence. The cells from the T-150 were recovered by trypsinization and cryopreserved at passage number 145. The cells from the T-25 were recovered by trypsinization every 3 days to 4 days and maintained up to passage 153. The cells were induced as above and samples retested by p24 ELISA at two different passages prior to passage 153.

[0069] The two highest p24 producers were chosen and were recovered by trypsinization every 3 days to 4 days up to passage 163. Samples from the clones were tested by p24 and gp120 ELISA from passage 158 and by p24 ELISA at passage 163, to assess clonal stability. The most suitable of these two cell lines, named 148 to 391, was chosen for further sub-cloning. The clone nomenclature defines the experiment number for this procedure, which was 148, and the number of the clone, which was number 391 of the original 394 isolated.

[0070] The vero cells were grown for approximately 100 h to 103 h and the medium was then replaced with growth medium containing 5-azacytidine. The bottles were then incubated for a further 20 h to 22 h, at which time the medium was replaced with serum-free medium containing CdCl₂ and ZnCl₂. The bottles were then incubated for 29 h to 31 h, at which time the medium was harvested, pooled and stored at 2° C. to 8° C. prior to purification.

[0071] The next day after harvesting, the solution was clarified, concentrated and diafiltered against phosphate buffer. The concentrate was passed through a ceramic hydroxyapatite (type I) column and the run-through was collected. The run-through from two successive sublots was pooled together and pumped onto a sucrose density gradient in a continuous zonal ultracentrifuge rotor. Pseudovirion-containing fractions were collected and pooled. The pooled pseudovirion fractions were diafiltered against PBS containing 2.5% sucrose to reduce the sucrose content, concentrated and diafiltered again. The material was sterile filtered using a 0.2 μm filter. At this stage the materials was designated as a purified sub-lot and were stored at 2 to 8° C.

[0072] The adjuvants were prepared separately and filter sterilized before filling in single dose vials. QS21 was stored at −20° C.

Example 4

[0073] This Example describes the production of recombinant pox virus vCP1579.

[0074] Recombinant pox virus vCP1579 (FIG. 4) contains the HIV-1 gag and protease genes derived from the HIV-1 IIIB isolate, the gp120 envelope sequences derived from the HIV-1 Bx08 isolate, and sequences encoding a polypeptide encompassing the known human CTL epitopes from HIV-1 Nef and Pol.

[0075] Recombinant vCP1579 (FIG. 4) was generated by insertion of the vector modifying sequences from pMPC6H6K3E3 (FIG. 8) encoding E3L and K3L into the C6 site of recombinant vCP1566 (FIG. 4). Recombinant vCP1566 was generated by insertion of an expression cassette encoding a synthetic polypeptide containing Pol CTL epitopes and Nef CTL epitopes (FIG. 11) and plasmid pMPC5H6PN (FIG. 9) into vCP1453 at the insertion site known as C5. Recombinant vCP1453 was generated by co-insertion of genes encoding HIV-1 env and gag/protease gene products, plasmid pHIV76 (FIG. 10), into the ALVAC genome at the insertion site known as C3.

[0076] The construction of recombinant pox vectors containing the E3L and K3L genes has been described in U.S. Pat. No. 6,004,777 issued Dec. 21, 1999 to Tartaglia et al. and the recombinant pox vectors describing the insertion of HIV genes has been described in U.S. Pat. No. 5,766,598 issued Jun. 16, 1998 to Paoletti et al.

[0077] The locus designated C3 was used for the insertion of the HIV-1 env and gag gene sequences into the ALVAC(2) vector, and the locus designated as C5 was the insertion site for the sequences encoding the HIV-1 Nef and Pol CTL epitopes. By virtue of the C3 and C5 loci existing within the extensive inverted terminal repetitions (ITRs) of the virus genome (approximately 41 kbp), insertion into these loci results in the occurrence of two copies of the inserted HIV-1 sequences.

[0078] Briefly, expression cassette pHIV76 (FIG. 10) was engineered in the following manner. Plasmid p133B1 (FIG. 3) containing the HIV-1Bx08 gp 160 gene was used as the starting plasmid. The 3′-end of the H6 promoter was cloned upstream of the gp160 gene and three poxvirus early transcription termination signal sequences (T₅NT) were modified. This was accomplished by cloning a 2,600 bp BamHI-digested PCR fragment, containing the 3′-end of the H6 promoter and the T₅NT-modified HIV-1 (BX08) gp160 gene, into the BamHI site of pBS-SK. This PCR fragment was generated from four overlapping PCR fragments (a 570 bp fragment, a 140 bp fragment, a 500 bp fragment and a 1,450 bp fragment) and the oligonucleotides, RW835 (5′-ATCATCATCGGATCC CGGGGTCGCGATATCCGTTAAGTTTGTATCGTAATGAAAGTGAAGGAC C-3′-SEQ ID NO: 2) and RW836 (5′-ATCATCATCGGATCCCGGGGTT ATAGCAAAGCCCTTTC-3′-SEQ ID NO: 3). The 570 bp PCR fragment, containing the 3′-end of the H6 promoter and the 5′-end of the gp160 gene, was generated from the plasmid, p133B1, with the oligonucleotides, RW835 (5′-ATC ATCATCGGATCCCGGGGTCGCGATATCCGTTAAGTTTGTATCGTAATG AAAGTGAAGGAGACC-3′) and RW868 (5′-ATCAAGACTATAGAAGA GTGCATATTCTCTCTTCATC-3′). The 140 bp PCR fragment, containing an interior portion of the gp160 gene, was generated from plasmid p133-B1 with the oligonucleotides, RW864 (5′-GCACTCTTCTATAGTCTTGATATAGTAC-3′-SEQ ID NO: 4) and RW865 (5′-AGCCGGGGCGCAGAAATGTATG GGAATTGGCAC-3′-SEQ ID NO: 5). The 500 bp PCR fragment, containing an interior portion of the gp160 gene, was generated from 133-3 with the oligonucleotides, RW866 (5′-ATACATTTCTGCGCCCCGGCTGGT TTTGCGATTC-3′-SEQ ID NO: 6) and RW867 (5′-GAAGAATTC CCCTCCACAATTAAAAC-3′-SEQ ID NO: 7). The 1,450 bp PCR fragment, containing the 3′-end of the gp160 gene, was generated from p133-B1 with the oligonucleotides, RW869 (5′-TGTGGAGGGGAATTCTTCTACTGTAATAC AACACAAC-3′-SEQ ID NO: 8) and RW836 (5′-ATCATCATCGGAT CCCGGGGTTATAGCAAAGCCCTTTC-3′-SEQ ID NO: 9). The 3′-end of the 570 bp PCR fragment overlaps the 5′-end of the 140 bp PCR fragment. The 3′-end of the 140 bp PCR fragment overlaps the 5′-end of the 500 bp PCR fragment. The 3′-end of the 500 bp PCR fragment overlaps the 5′-end of the 1450 bp PCR fragment. The plasmid generated by this manipulation is called pRW997.

[0079] The sequence encoding gp41 was then replaced with the sequence encoding the gp160 transmembrane (TM) region. This modification was accomplished by cloning a 200 bp MfeI-HindIII-digested PCR fragment, containing the 3′-end of the gp120 gene and the TM sequence, into the 4,400 bp MfeI-HindIII fragment of pRW997. This PCR fragment was generated from two overlapping PCR fragments (a 170 bp fragment and a 125 bp fragment) with the oligonucleotides, HIVP97 (5′-TAGTGGGAAAGAGATCTTCAGACC-3′-SEQ ID NO: 10) and HIVP101 (5′-TTTTAAGCTTTTATCCCTGCCTAACT CTATTCAC TAT-3′-SEQ ID NO: 11). The 170 bp PCR fragment was generated from pRW997 with the oligonucleotides, HIP97 (5′-TAGTGGGAAAGAGATCTTCAGACC-3′-SEQ ID NO: 12) and HIVP100 (5′-CCTCCTACTATCATTATGAATATTCTTTTTTCTCTCTGCACCACTCT-3′-SEQ ID NO: 13). The 125 bp PCR fragment was generated from pRW997 with the oligonucleotides, HIVP99 (5′-AGAGTGGTGCAGAGAGAAAAA AGAATATTCATAATGATAGTAGGAGGC-3′-SEQ ID NO: 14) and HIVP101 (5′-TTTTAAGCTTTTA TCCCTGCCTAACTCTATTCACTAT-3′-SEQ ID NO: 15). The plasmid generated by this manipulation is called pHIV71.

[0080] The H6-promoted gp120+TM gene was then cloned between C3 flanking arms, into a plasmid containing the I3L-promoted HIV1 gag/(pro) gene. This modification was accomplished by cloning the 1,600 bp NruI-XhoI fragment of pHIV71, containing the H6-promoted gp120+TM gene, into the 8,200 bp NruI-XhoI fragment of pHIV63. The plasmid generated by this manipulation is called pHIV76 (FIG. 10). Plasmid pHIV76 was used in in vivo recombination experiments with ALVAC (CPpp) as rescue virus to yield vCP1453.

[0081] The sequence of the nef/pol regions is shown in FIG. 12 and the E3L and K3L sequences are shown in FIG. 13. To generate ALVAC(2)120(BX08)GNP (vCP1579), expression cassettes consisting of the promoter/HIV-1 gene combinations were subcloned into an ALVAC donor plasmid, which were then used to insert the expression cassettes into defined sites in the ALVAC genome by in vitro recombination as previously described (Ref 20).

Example 5

[0082] This Example describes the results of immunization regimes.

[0083] Groups of four animals (macaques) each were randomly assigned to seven vaccine groups as illustrated in Table 1. In this Table, “BX08 DNA” refers to pCMV3BX08, prepared as described in Example 1, “BX08 VLP” refers to the pseudovirions produced by expression vector p133B1in Vero cells, as described in Example 3, and “ALVAC(2) BX08” refers vCP1579, prepared as described in Example 4. Reference (pre-bleed) sera were sampled at −6 and −2 weeks pre-vaccination. Primary immunizations with the various vaccines were given on weeks 0 and 4 with boosts on weeks 24 and 44 (FIGS. 5A, 5B). The vaccines were immunized intramuscularly into one quadricep of each macaque monkey.

[0084] Sera were prepared from whole-blood using SST collection tubes and analyzed using commercially available HIV-1 western blots. Groups 1, 2 and 7 showed low levels of anti-Env antibodies after the first boost (FIGS. 6 and 7). Based on ELISA values, the anti-env antibody levels were below 1 μg/ml of specific IgG. High levels of anti-gag antibodies were detected in groups 1, 2, 3, 4, and 7 (FIGS. 6 and 7). No HIV-1 specific antibodies were detected in groups 5 and 6 (FIG. 6).

[0085] The ability of the antibodies raised in the immunized monkeys to neutralize HIV-1BX08 virus in human PBMC was assayed based on the reduction of p24 levels.

[0086] The neutralization assay was performed essentially as described in reference 18. Briefly, serum dilutions were mixed with HIV-1 BX08 and the mixtures incubated for 1 hour, then added to susceptible human PBMC cells. Titres were recorded as the dilution of serum at which p24 was reduced by 80%. Serum samples were assayed at 1:2, 1:8 and 1:32 dilution on the virus (1:6, 1:24 and 1:26 dilutions after the addition of cells). p24 levels were evaluated by p24-specific ELISA assay.

[0087] DNA vaccination on its own, group 5, and ALVAC on its own, group 6, had no monkeys showing reduction of p24 levels greater than 80%. The low DNA (600 ug) plus ALVAC, group 4, also showed no monkeys with greater than 80% reduction of p24 titres. VLP plus DNA, either high or low dose (group 1 and 2) showed enhanced reduction of p24 levels compared to VLPs alone, group 7. High dose DNA, group 3, in combination with ALVAC enhanced the ability to elicit p24 or virus neutralising antibodies over the low dose, group 4 or ALVAC alone, group 6. These results indicate that DNA vaccination in combination with VLPs or ALVAC enhanced the levels of virus neutralising antibodies as indicated by the reduction of p24 levels in the sera of the immunized monkeys.

[0088] The percentage reduction of p24 is calculated relative to the amount of p24 produced in the presence of the corresponding dilution of week 2 samples.

[0089] SUMMARY OF DISCLOSURE

[0090] In summary of this disclosure, the present invention provides novel immunization procedures and immunogenic compositions for generating virus neutralizing levels of antibodies to a primary HIV isolate and vectors utilized therein and for the generation of components for use therein. Modifications are possible within the scope of this invention. TABLE 1 Study Design Group number Treatment - Week 0, 4 Treatment - Week 24, 44 1  3 mg BX08 DNA  3 mg BX08 DNA  50 μg BX08 VLP  50 μg BX08 VLP 100 μg QS21 100 μg QS21 2 600 μg BX08 DNA 600 μg BX08 DNA  50 μg BX08 VLP  50 μg BX08 VLP 100 μg QS21 100 μg QS21 3  3 mg BX08 DNA ALVAC(2) BX08 (1 × 10⁸ pfu) 4 600 μg BX08 DNA ALVAC(2) BX08 (1 × 10⁸ pfu) 5  3 mg BX08 DNA  3 mg BX08 DNA 6 Control DNA ALVAC(2) BX08 (1 × 10⁸ pfu) 7  50 μg BX08 VLP  50 μg BX08 VLP 100 μg QS21 100 μg QS21

[0091] TABLE 2 Number of Monkeys showing > 80% reduction of p24 titre. Group number Week 26 Bleed Week 44 Bleed 1 3/4 ¾ 2 3/4 4/4 3 2/4 2/4 4 0/4 0/4 5 0/4 0/4 6 0/4 0/4 7 2/4 3/4

[0092] References

[0093] 1. UNAIDS, WHO. AIDS epidemic update: December 1999. Geneva: World Health Organisation; 1999.

[0094] 2. Heyward et al 1998. HIV vaccine development and evaluation: realistic expectations. AIDS Res Hum Retrivir 14:S205-S210.

[0095] 3. Haigwood N L and Zolla-Pazner S. 1998. Humoral immunity to HIV, SIV and SHIV. AIDS 12: S121-S132.

[0096] 4. Johnson et al. 1998. Cellular immune responses to HIV-1. AIDS 12:S113-120.

[0097] 5. Keefer, M. et al 1996. AIDS Res Hum Retroviruses. 12:683-693.

[0098] 6. Cox W. et al 1993. Virology 195:845-850.

[0099] 7. Graham B S, Keefer M C, McElrath M J, Gorse G J, Schwartz D H, Weinhold K, Matthews T J, Esterlitz J R, Sinangil F, Fast P E. 1996. Ann Intern Med August 15;125(4):270-9.

[0100] 8. Pincus S H, Messer K G, Cole R, Ireland R, VanCott T C, Pinter A, Schwartz D H, Graham B S, Gorse G J. 1997. J Immunol 1997 April 1;158(7):3511-20.

[0101] 9. Fang Z Y et al. 1999. J Infect Dis. 180(4):1122-32.

[0102] 10. Rovinski B et al. 1995. AIDS Res Hum Retroviruses. 11:1187-1195.

[0103] 11. Arp J. et al. 1999. Viral Immunolgy 12(4):281-296.

[0104] 12. Zolka-Pazner et al, J. Virology, vol. 72:1052-1059, 1998.

[0105] 13. Karin M and Richards R I. Human metallothionein genes—primary structure of the metallothionein-II gene and a related processed gene. Nature 1982;299:797-802.

[0106] 14. Sambrook J et al. Molecular Cloning A Laboratory Manual, Second Ed.: Cold Spring Harbour Laboratory Press. 1989.

[0107] 15. Haynes J R, Cao S X, Rovinski B, Sia C, James O, Dekaban G A, Klein M H. Production of immunogenic HIV-1 viruslike particles in stably engineered monkey cell lines. AIDS Res Hum Retroviruses 1991;7:17-27.

[0108] 16. Persson R, Cao S, Cates G, Yao F, Klein M, Rovinsid B. Modifications of HIV-1 Retrovirus-like Particles to Enhance Safety and Immunogenicity. Biologicals 1998, 26(4): 255-265.

[0109] 17. Southern P J and Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Molec Appl Genet 1982;1: 327-341.

[0110] 18. Graham, B. S. et al, 1993, J. Infection Des. 167:533-537.

[0111] 19. Tartaglia, J. et al., 1992, Virology, 188:219-232.

[0112] 20. Piccini, A. et al., 1987, Methods in Enzymology 153:545-563.

1 19 1 1345 DNA Human immunodeficiency virus 1 tttttttcat tatttagaaa ttatgcattt tagatcttta taagcggccg tgattaacta 60 gtcataaaaa cccgggatcg attctagact cgagggtacc ggatcttaat taattagtca 120 tcaggcaggg cgagaacgag actatctgct cgttaattaa ttaggtcgac ggatccccca 180 acaaaaacta atcagctatc ggggttaatt aattagttat tagacaaggt gaaaacgaaa 240 ctatttgtag cttaattaat tagagcttct ttattctata cttaaaaagt gaaaataaat 300 acaaaggttc ttgagggttg tgttaaattg aaagcgagaa ataatcataa attatttcat 360 tatcgcgata tccgttaagt ttgtatcgta atgccactaa cagaagaagc agagctagaa 420 ctggcagaaa acagagagat tctaaaagaa ccagtacatg gagtgtatta tgacccatca 480 aaagacttaa tagcagaaat acagaagcag gggcaaggcc aatggacata tcaaatttat 540 caagagccat ttaaaaatct gaaaacagga atggagtgga gatttgattc tagattagca 600 tttcatcacg tagctagaga attacatcct gaatatttta aaaattgtat ggcaatattc 660 caaagtagca tgacaaaaat cttagagcct tttagaaaac aaaatccaga catagttatc 720 tatcaataca tggatgattt gtatgtagga tctgacttag aaatagggca gcatagaaca 780 aaaatagagg agctgagaca acatctgttg aggtggggac ttacaaccat ggtaggtttt 840 ccagtaacac ctcaagtacc tttaagacca atgacttaca aagcagctgt agatctttct 900 cactttttaa aagaaaaagg aggtttagaa gggctaattc attctcaacg aagacaagat 960 attcttgatt tgtggattta tcatacacaa ggatattttc ctgattggca gaattacaca 1020 ccaggaccag gagtcagata cccattaacc tttggttggt gctacaagct agtaccaatg 1080 attgagactg taccagtaaa attaaagcca ggaatggatg gcccaaaagt taaacaatgg 1140 ccattgacag aagaaaaaat aaaagcatta gtagaaattt gtacagagat ggaaaaggaa 1200 gggaaaattt caaaaattgg gccttaattt ttctgcagcc cgggggatcc tttttatagc 1260 taattagtca cgtacctttg agagtaccac ttcagctacc tcttttgtgt ctcagagtaa 1320 ctttctttaa tcaattccaa aacag 1345 2 64 DNA Human immunodeficiency virus 2 atcatcatcg gatcccgggg tcgcgatatc cgttaagttt gtatcgtaat gaaagtgaag 60 gacc 64 3 38 DNA Human immunodeficiency virus 3 atcatcatcg gatcccgggg ttatagcaaa gccctttc 38 4 28 DNA Human immunodeficiency virus 4 gcactcttct atagtcttga tatagtac 28 5 33 DNA Human immunodeficiency virus 5 agccggggcg cagaaatgta tgggaattgg cac 33 6 34 DNA Human immunodeficiency virus 6 atacatttct gcgccccggc tggttttgcg attc 34 7 26 DNA Human immunodeficiency virus 7 gaagaattcc cctccacaat taaaac 26 8 37 DNA Human immunodeficiency virus 8 tgtggagggg aattcttcta ctgtaataca acacaac 37 9 38 DNA Human immunodeficiency virus 9 atcatcatcg gatcccgggg ttatagcaaa gccctttc 38 10 24 DNA Human immunodeficiency virus 10 tagtgggaaa gagatcttca gacc 24 11 37 DNA Human immunodeficiency virus 11 ttttaagctt ttatccctgc ctaactctat tcactat 37 12 24 DNA Human immunodeficiency virus 12 tagtgggaaa gagatcttca gacc 24 13 47 DNA Human immunodeficiency virus 13 cctcctacta tcattatgaa tattcttttt tctctctgca ccactct 47 14 48 DNA Human immunodeficiency virus 14 agagtggtgc agagagaaaa aagaatattc ataatgatag taggaggc 48 15 37 DNA Human immunodeficiency virus 15 ttttaagctt ttatccctgc ctaactctat tcactat 37 16 4434 DNA Human immunodeficiency virus 16 gagctcgcgg ccgcctatca aaagtcttaa tgagttaggt gtagatagta tagatattac 60 tacaaaggta ttcatatttc ctatcaattc taaagtagat gatattaata actcaaagat 120 gatgatagta gataatagat acgctcatat aatgactgca aatttggacg gttcacattt 180 taatcatcac gcgttcataa gtttcaactg catagatcaa aatctcacta aaaagatagc 240 cgatgtattt gagagagatt ggacatctaa ctacgctaaa gaaattacag ttataaataa 300 tacataatgg attttgttat catcagttat atttaacata agtacaataa aaagtattaa 360 ataaaaatac ttacttacga aaaaatgact aattagctat aaaaacccag atctctcgag 420 gtcgacggta tcgataagct tgatatcgaa ttcataaaaa ttattgatgt ctacacatcc 480 ttttgtaatt gacatctata tatccttttg tataatcaac tctaatcact ttaactttta 540 cagttttccc taccagttta tccctatatt caacatatct atccatatgc atcttaacac 600 tctctgccaa gatagcttca gagtgaggat agtcaaaaag ataaatgtat agagcataat 660 ccttctcgta tactctgccc tttattacat cgcccgcatt gggcaacgaa taacaaaatg 720 caagcatacg atacaaactt aacggatatc gcgataatga aataatttat gattatttct 780 cgctttcaat ttaacacaac cctcaagaac ctttgtattt attttcactt tttaagtata 840 gaataaagaa agctctaatt aattaatgaa cagattgttt cgttttcccc ttggcgtatc 900 actaattaat taacccgggc tgcagctcga ggaattcaac tatatcgaca tatttcattt 960 gtatacacat aaccattact aacgtagaat gtataggaag agatgtaacg ggaacagggt 1020 ttgttgattc gcaaactatt ctaatacata attcttctgt taatacgtct tgcacgtaat 1080 ctattataga tgccaagata tctatataat tattttgtaa gatgatgtta actatgtgat 1140 ctatataagt agtgtaataa ttcatgtatt tcgatatatg ttccaactct gtctttgtga 1200 tgtctagttt cgtaatatct atagcatcct caaaaaatat attcgcatat attcccaagt 1260 cttcagttct atcttctaaa aaatcttcaa cgtatggaat ataataatct attttacctc 1320 ttctgatatc attaatgata tagtttttga cactatcttc tgtcaattga ttcttattca 1380 ctatatctaa gaaacggata gcgtccctag gacgaactac tgccattaat atctctatta 1440 tagcttctgg acataattca tctattatac cagaattaat gggaactatt ccgtatctat 1500 ctaacatagt tttaagaaag tcagaatcta agacctgatg ttcatatatt ggttcataca 1560 tgaaatgatc tctattgatg atagtgacta tttcattctc tgaaaattgg taactcattc 1620 tatatatgct ttccttgttg atgaaggata gaatatactc aatagaattt gtaccaacaa 1680 actgttctct tatgaatcgt atatcatcat ctgaaataat catgtaaggc atacatttaa 1740 caattagaga cttgtctcct gttatcaata tactattctt gtgataattt atgtgtgagg 1800 caaatttgtc cacgttcttt aattttgtta tagtagatat caaatccaat ggagctacag 1860 ttcttggctt aaacagatat agtttttctg gaacaaattc tacaacatta ttataaagga 1920 ctttgggtag ataagtggga tgaaatccta ttttaattaa tgctatcgca ttgtcctcgt 1980 gcaaatatcc aaacgctttt gtgatagtat ggcattcatt gtctagaaac gctctacgaa 2040 tatctgtgac agatatcatc tttagagaat atactagtcg cgttaatagt actacaattt 2100 gtatttttta atctatctca ataaaaaaat taatatgtat gattcaatgt ataactaaac 2160 tactaactgt tattgataac tagaatcaga atctaatgat gacgtaacca agaagtttat 2220 ctactgccaa tttagctgca ttatttttag catctcgttt agattttcca tctgccttat 2280 cgaatactct tccgtcgatg tctacacagg cataaaatgt aggagagtta ctaggcccaa 2340 ctgattcaat acgaaaagac caatctctct tagttatttg gcagtactca ttaataatgg 2400 tgacagggtt agcatctttc caatcaataa tttttttagc cggaataaca tcatcaaaag 2460 acttatgatc ctctctcatt gatttttcgc gggatacatc atctattatg acgtcagcca 2520 tagcatcagc atccggctta tccgcctccg ttgtcataaa ccaacgagga ggaatatcgt 2580 cggagctgta caccatagca ctacgttgaa gatcgtacag agctttatta acttctcgct 2640 tctccatatt aagttgtcta gttagttgtg cagcagtagc tccttcgatt ccaatgtttt 2700 taatagccgc acacacaatc tctgcgtcag aacgctcgtc aatatagatc ttagacattt 2760 ttagagagaa ctaacacaac cagcaataaa actgaaccta ctttatcatt tttttattca 2820 tcatcctctg gtggttcgtc gtttctatcg aatgtagctc tgattaaccc gtcatctata 2880 ggtgatgctg gttctggaga ttctggagga gatggattat tatctggaag aatctctgtt 2940 atttccttgt tttcatgtat cgattgcgtt gtaacattaa gattgcgaaa tgctctaaat 3000 ttgggaggct taaagtgttg tttgcaatct ctacacgcgt gtctaactag tggaggttcg 3060 tcagctgctc tagtttgaat catcatcggc gtagtattcc tacttttaca gttaggacac 3120 ggtgtattgt atttctcgtc gagaacgtta aaataatcgt tgtaactcac atcctttatt 3180 ttatctatat tgtattctac tcctttctta atgcatttta taccgaataa gagatagcga 3240 aggaattctt tttattgatt aactagtcaa atgagtatat ataattgaaa aagtaaaata 3300 taaatcatat aataatgaaa cgaaatatca gtaatagaca ggaactggca gattcttctt 3360 ctaatgaagt aagtactgct aaatctccaa aattagataa aaatgataca gcaaatacag 3420 cttcattcaa cgaattacct tttaattttt tcagacacac cttattacaa actaactaag 3480 tcagatgatg agaaagtaaa tataaattta acttatgggt ataatataat aaagattcat 3540 gatattaata atttacttaa cgatgttaat agacttattc catcaacccc ttcaaacctt 3600 tctggatatt ataaaatacc agttaatgat attaaaatag attgtttaag agatgtaaat 3660 aattatttgg aggtaaagga tataaaatta gtctatcttt cacatggaaa tgaattacct 3720 aatattaata attatgatag gaatttttta ggatttacag ctgttatatg tatcaacaat 3780 acaggcagat ctatggttat ggtaaaacac tgtaacggga agcagcattc tatggtaact 3840 ggcctatgtt taatagccag atcattttac tctataaaca ttttaccaca aataatagga 3900 tcctctagat atttaatatt atatctaaca acaacaaaaa aatttaacga tgtatggcca 3960 gaagtatttt ctactaataa agataaagat agtctatctt atctacaaga tatgaaagaa 4020 gataatcatt tagtagtagc tactaatatg gaaagaaatg tatacaaaaa cgtggaagct 4080 tttatattaa atagcatatt actagaagat ttaaaatcta gacttagtat aacaaaacag 4140 ttaaatgcca atatcgattc tatatttcat cataacagta gtacattaat cagtgatata 4200 ctgaaacgat ctacagactc aactatgcaa ggaataagca atatgccaat tatgtctaat 4260 attttaactt tagaactaaa acgttctacc aatactaaaa ataggatacg tgataggctg 4320 ttaaaagctg caataaatag taaggatgta gaagaaatac tttgttctat accttcggag 4380 gaaagaactt tagaacaact taagtttaat caaacttgta tttatgaagg tacc 4434 17 4434 DNA Human immunodeficiency virus 17 ctcgagcgcc ggcggatagt tttcagaatt actcaatcca catctatcat atctataatg 60 atgtttccat aagtataaag gatagttaag atttcatcta ctataattat tgagtttcta 120 ctactatcat ctattatcta tgcgagtata ttactgacgt ttaaacctgc caagtgtaaa 180 attagtagtg cgcaagtatt caaagttgac gtatctagtt ttagagtgat ttttctatcg 240 gctacataaa ctctctctaa cctgtagatt gatgcgattt ctttaatgtc aatatttatt 300 atgtattacc taaaacaata gtagtcaata taaattgtat tcatgttatt tttcataatt 360 tatttttatg aatgaatgct tttttactga ttaatcgata tttttgggtc tagagagctc 420 cagctgccat agctattcga actatagctt aagtattttt aataactaca gatgtgtagg 480 aaaacattaa ctgtagatat ataggaaaac atattagttg agattagtga aattgaaaat 540 gtcaaaaggg atggtcaaat agggatataa gttgtataga taggtatacg tagaattgtg 600 agagacggtt ctatcgaagt ctcactccta tcagtttttc tatttacata tctcgtatta 660 ggaagagcat atgagacggg aaataatgta gcgggcgtaa cccgttgctt attgttttac 720 gttcgtatgc tatgtttgaa ttgcctatag cgctattact ttattaaata ctaataaaga 780 gcgaaagtta aattgtgttg ggagttcttg gaaacataaa taaaagtgaa aaattcatat 840 cttatttctt tcgagattaa ttaattactt gtctaacaaa gcaaaagggg aaccgcatag 900 tgattaatta attgggcccg acgtcgagct ccttaagttg atatagctgt ataaagtaaa 960 catatgtgta ttggtaatga ttgcatctta catatccttc tctacattgc ccttgtccca 1020 aacaactaag cgtttgataa gattatgtat taagaagaca attatgcaga acgtgcatta 1080 gataatatct acggttctat agatatatta ataaaacatt ctactacaat tgatacacta 1140 gatatattca tcacattatt aagtacataa agctatatac aaggttgaga cagaaacact 1200 acagatcaaa gcattataga tatcgtagga gttttttata taagcgtata taagggttca 1260 gaagtcaaga tagaagattt tttagaagtt gcatacctta tattattaga taaaatggag 1320 aagactatag taattactat atcaaaaact gtgatagaag acagttaact aagaataagt 1380 gatatagatt ctttgcctat cgcagggatc ctgcttgatg acggtaatta tagagataat 1440 atcgaagacc tgtattaagt agataatatg gtcttaatta cccttgataa ggcatagata 1500 gattgtatca aaattctttc agtcttagat tctggactac aagtatataa ccaagtatgt 1560 actttactag agataactac tatcactgat aaagtaagag acttttaacc attgagtaag 1620 atatatacga aaggaacaac tacttcctat cttatatgag ttatcttaaa catggttgtt 1680 tgacaagaga atacttagca tatagtagta gactttatta gtacattccg tatgtaaatt 1740 gttaatctct gaacagagga caatagttat atgataagaa cactattaaa tacacactcc 1800 gtttaaacag gtgcaagaaa ttaaaacaat atcatctata gtttaggtta cctcgatgtc 1860 aagaaccgaa tttgtctata tcaaaaagac cttgtttaag atgttgtaat aatatttcct 1920 gaaacccatc tattcaccct actttaggat aaaattaatt acgatagcgt aacaggagca 1980 cgtttatagg tttgcgaaaa cactatcata ccgtaagtaa cagatctttg cgagatgctt 2040 atagacactg tctatagtag aaatctctta tatgatcagc gcaattatca tgatgttaaa 2100 cataaaaaat tagatagagt tattttttta attatacata ctaagttaca tattgatttg 2160 atgattgaca ataactattg atcttagtct tagattacta ctgcattggt tcttcaaata 2220 gatgacggtt aaatcgacgt aataaaaatc gtagagcaaa tctaaaaggt agacggaata 2280 gcttatgaga aggcagctac agatgtgtcc gtattttaca tcctctcaat gatccgggtt 2340 gactaagtta tgcttttctg gttagagaga atcaataaac cgtcatgagt aattattacc 2400 actgtcccaa tcgtagaaag gttagttatt aaaaaaatcg gccttattgt agtagttttc 2460 tgaatactag gagagagtaa ctaaaaagcg ccctatgtag tagataatac tgcagtcggt 2520 atcgtagtcg taggccgaat aggcggaggc aacagtattt ggttgctcct ccttatagca 2580 gcctcgacat gtggtatcgt gatgcaactt ctagcatgtc tcgaaataat tgaagagcga 2640 agaggtataa ttcaacagat caatcaacac gtcgtcatcg aggaagctaa ggttacaaaa 2700 attatcggcg tgtgtgttag agacgcagtc ttgcgagcag ttatatctag aatctgtaaa 2760 aatctctctt gattgtgttg gtcgttattt tgacttggat gaaatagtaa aaaaataagt 2820 agtaggagac caccaagcag caaagatagc ttacatcgag actaattggg cagtagatat 2880 ccactacgac caagacctct aagacctcct ctacctaata atagaccttc ttagagacaa 2940 taaaggaaca aaagtacata gctaacgcaa cattgtaatt ctaacgcttt acgagattta 3000 aaccctccga atttcacaac aaacgttaga gatgtgcgca cagattgatc acctccaagc 3060 agtcgacgag atcaaactta gtagtagccg catcataagg atgaaaatgt caatcctgtg 3120 ccacataaca taaagagcag ctcttgcaat tttattagca acattgagtg taggaaataa 3180 aatagatata acataagatg aggaaagaat tacgtaaaat atggcttatt ctctatcgct 3240 tccttaagaa aaataactaa ttgatcagtt tactcatata tattaacttt ttcattttat 3300 atttagtata ttattacttt gctttatagt cattatctgt ccttgaccgt ctaagaagaa 3360 gattacttca ttcatgacga tttagaggtt ttaatctatt tttactatgt cgtttatgtc 3420 gaagtaagtt gcttaatgga aaattaaaaa agtctgtgtg gaataatgtt tgattgattc 3480 agtctactac tctttcattt atatttaaat tgaataccca tattatatta tttctaagta 3540 ctataattat taaatgaatt gctacaatta tctgaataag gtagttgggg aagtttggaa 3600 agacctataa tattttatgg tcaattacta taattttatc taacaaattc tctacattta 3660 ttaataaacc tccatttcct atattttaat cagatagaaa gtgtaccttt acttaatgga 3720 ttataattat taatactatc cttaaaaaat cctaaatgtc gacaatatac atagttgtta 3780 tgtccgtcta gataccaata ccattttgtg acattgccct tcgtcgtaag ataccattga 3840 ccggatacaa attatcggtc tagtaaaatg agatatttgt aaaatggtgt ttattatcct 3900 aggagatcta taaattataa tatagattgt tgttgttttt ttaaattgct acataccggt 3960 cttcataaaa gatgattatt tctatttcta tcagatagaa tagatgttct atactttctt 4020 ctattagtaa atcatcatcg atgattatac ctttctttac atatgttttt gcaccttcga 4080 aaatataatt tatcgtataa tgatcttcta aattttagat ctgaatcata ttgttttgtc 4140 aatttacggt tatagctaag atataaagta gtattgtcat catgtaatta gtcactatat 4200 gactttgcta gatgtctgag ttgatacgtt ccttattcgt tatacggtta atacagatta 4260 taaaattgaa atcttgattt tgcaagatgg ttatgatttt tatcctatgc actatccgac 4320 aattttcgac gttatttatc attcctacat cttctttatg aaacaagata tggaagcctc 4380 ctttcttgaa atcttgttga attcaaatta gtttgaacat aaatacttcc atgg 4434 18 88 PRT Human immunodeficiency virus 18 Gln His Arg Cys Met Arg Lys Tyr Asn Val Asp Ile Tyr Gly Lys Thr 1 5 10 15 Tyr Asp Val Arg Ile Val Lys Val Lys Val Thr Lys Gly Val Leu Lys 20 25 30 Asp Arg Tyr Glu Val Tyr Arg Asp Met His Met Lys Val Ser Glu Ala 35 40 45 Leu Ile Ala Glu Ser His Pro Tyr Asp Phe Leu Tyr Ile Tyr Leu Ala 50 55 60 Tyr Asp Lys Glu Tyr Val Arg Gly Lys Ile Val Asp Gly Ala Asn Pro 65 70 75 80 Leu Ser Tyr Cys Phe Ala Leu Met 85 19 190 PRT Human immunodeficiency virus 19 Phe Arg Ile Ile Val Tyr Gly Leu Leu Lys Asp Val Ala Leu Lys Ala 1 5 10 15 Ala Asn Asn Lys Ala Asp Arg Lys Ser Lys Gly Asp Ala Lys Asp Phe 20 25 30 Val Arg Gly Asp Ile Asp Val Cys Ala Tyr Phe Thr Pro Ser Asn Ser 35 40 45 Pro Gly Val Ser Glu Ile Arg Phe Ser Trp Asp Arg Lys Thr Ile Gln 50 55 60 Cys Tyr Glu Asn Ile Ile Thr Val Pro Asn Ala Asp Lys Trp Asp Ile 65 70 75 80 Ile Lys Lys Ala Pro Ile Val Asp Asp Phe Ser Lys His Asp Glu Arg 85 90 95 Met Ser Lys Glu Arg Ser Val Asp Asp Ile Ile Val Asp Ala Met Ala 100 105 110 Asp Ala Asp Pro Lys Asp Ala Glu Thr Thr Met Phe Trp Arg Pro Pro 115 120 125 Ile Asp Asp Ser Ser Tyr Val Met Ala Ser Arg Gln Leu Asp Tyr Leu 130 135 140 Ala Lys Asn Val Glu Arg Lys Glu Met Asn Leu Gln Arg Thr Leu Gln 145 150 155 160 Ala Ala Thr Ala Gly Glu Ile Gly Ile Asn Lys Ile Ala Ala Cys Val 165 170 175 Ile Glu Ala Asp Ser Arg Glu Asp Ile Tyr Ile Lys Ser Met 180 185 190 

What we claim is:
 1. A method for generating in a host a virus neutralizing level of antibodies to a primary HIV isolate, comprising: at least one administration of a priming antigen to the host, wherein the priming antigen comprises a DNA molecule encoding an envelope glycoprotein of a primary isolate of HIV-1, resting the host for at least one specific resting period to provide for clonal expansion of an HIV antigen specific population of precursor B-cells therein to provide a primed host, and at least one administration of a boosting antigen to the primed host to provide said neutralizing levels of antibodies, wherein the boosting antigen is selected from the group consisting of a non-infectious, non-replicating, immunogenic HIV-like particle having at least the envelope glycoprotein of a primary isolate of HIV-1 and an attenuated viral vector expressing at least an envelope glycoprotein of a primary isolate of HIV-1.
 2. The method of claim 1 wherein said primary isolate is Bx08.
 3. The method of claim 2 wherein said DNA molecule is contained in a plasmid vector under the control of a heterologous promoter for expression of the envelope glycoprotein in the host.
 4. The method of claim 3 wherein the promoter is the cytomegalovirus promoter.
 5. The method of claim 4 wherein the vector has the identifying characteristics of pCMV3Bx08 shown in FIG.
 2. 6. The method of claim 1 wherein the at least one administration of a priming antigen is at least two administrations of the priming antigen.
 7. The method of claim 6 wherein the at least one specific resting period is effected after each priming administration.
 8. The method of claim 1 wherein the at least one specific resting period is between about 2 months to about 12 months.
 9. The method of claim 1 wherein said non-infectious, non-replicating, immunogenic HIV-like particle comprises an assembly of: (i) an env gene product, (ii) a pol gene product, and (iii) a gag gene product, said particle being encoded by a modified HIV genome deficient in long terminal repeats (LTRs) and containing gag, pol and env in their natural genomic arrangement.
 10. The method of claim 9 wherein the env gene is that from primary isolate BX08.
 11. The method of claim 1 wherein said non-infectious, non-replicating, immunogenic HIV-like particle is administered in conjunction with an adjuvant.
 12. The method of claim 11 wherein the adjuvant is QS21.
 13. The method of claim 1 wherein said attenuated viral vector is an attenuated avipoxvirus
 14. The method of claim 13 wherein the attenuated viral vector contains a modified HIV-genome deficient in long terminal repeats, wherein at least the env gene is that from primary isolate BX08.
 15. The method of claim 14 wherein the attenuated avipoxviris vector is the attenuated canary poxvirus ALVAC.
 16. The method of claim 15 wherein the attenuated canary poxvirus vector has the identifying characteristics of vCP1579.
 17. The method of claim 1 wherein the at least one administration of a boosting antigen is at least two administrations of a boosting antigen.
 18. A vector, comprising a DNA sequence encoding an envelope glycoprotein of a primary isolate of HIV-1 under the control of a heterologous promoter for expression of the envelope glycoprotein in a host organism.
 19. The vector of claim 18 wherein the vector is a plasmid vector.
 20. The vector of claim 18 wherein said primary HIV-1 isolate is Bx08.
 21. The vector of claim 20 wherein the promoter is the cytomegalovirus promoter.
 22. The vector of claim 21 which has the identifying characteristics of pCMV3Bx08 shown in FIG.
 2. 23. The vector of claim 18 wherein the vector is an attenuated viral vector. 